Mammalian cells have evolved sophisticated DNA repair systems to correct mispaired or damaged bases and extrahelical loops. We have surprisingly found that the eukaryotic mismatch recognition complex, MSH2/MSH3, not only fails to act as a guardian of the genome, but also causes CAG expansion, the lethal mutation underlying Huntington's disease (HD). It is the overall aim of this renewal to build on these discoveries, and to dissect the paradoxical mechanism by which binding of a CAG hairpin converts functional MSH2/MSH3 into a defective machine that causes mutation and disease. Although the molecular details of MutSa function are still incompletely understood, it is clear that some form of conformational coupling between DNA recognition and the nucleotide binding sites plays a central role. Our preliminary data indicate that MSH2/MSH3 binds with high affinity to the CAG hairpin, but binding there inhibits its ATPase activity, alters nucleotide affinity, and prevents translocation along DNA relative to repair competent loops. In order to define how the disease-causing CAG-hairpin diverts the MSH2MSH3 protein from normal repair, it becomes imperative to define the DNA and nucleotide dependent properties of the repair-competent MSH2MSH3-DNA complexes. The biochemical characterization of MSH2/MSH3 has lagged far behind that of MSH2/MSH6, and many parameters are unknown. MSH2/MSH3 is different in the way it recognizes DNA, and has different lesion specificity, and the sites of nucleotide binding in MSH2 and MSH3 subunits have not yet been mapped. Despite key differences, the biochemical properties of MSH2/MSH3 have been primarily extrapolated from MutS and its mammalian homologue, MSH2/MSH6. We propose two specific aims that elucidate the mechanism of uncoupling and how dissociation of DNA binding and ATP hydrolysis in MSH2/MSH3 leads to CAG expansion and disease. In Aim 1, we create a dynamic system for visualizing DNA and nucleotide dynamics in MSH2/MSh3-CAG hairpin complexes. Using combined smFRET and a panel of biochemical methods, We establish a real time system to monitor the relationships between DNA bindings, nucleotide binding and translocation of MSH2/MSh3 on repair competent and repair deficient substrates. In Aim2, we use SAX analysis and limited proteolysis to probe the DNA-induced conformational changes imposed on MSH2/MSH3 by hairpins binding. We will test whether those conformational alterations determine template specificity and influence in protein interactions that divert MSH2/MSh3 function in MMR. These two aims integrate protein biochemistry, nucleotide dynamics, and conformational analysis with in vivo biology in primary animals cells reflecting the disease. These studies will not only provide insights into how mutations in the mismatch repair genes cause expansion and disease, but will also broaden our conceptual framework for DNA damage recognition.